Isolation and Identification of a New Phenylpropanoid Derivative from the Fruits of Pimpinella Haussknechtii Rech. f. & Riedl and GC-MS Analysis of the Essential Oil.

The chemical composition of the essential oil of fruits of Pimpinella haussknechtii. was studied by GC-MS. After GC-MS analysis, one unknown component (56.7%) was observed, which was not characterized in the GC-MS library. The essential oil of P.haussknechtii was injected to HPLC using YMC-Pak-Sil column (250 × 20 mm) with gradient system of hexane (A), and hexane: ethyl acetate, 9:1 (B) to yield the interested compound as a new phenylpropanoid derivative. Its structure was elucidated as 4-(prop-2-enyl)-phenyl-3'-methylbutanoate based on (13)C- and (1)H-NMR as well as 2D-NMR, IR and different MS spectra. In the essential oil analysis, thirty-six components, comprising 94.9% of the total oil, were identified. 4-(2-propenyl)-phenyl 3'-methylbutyrate (56.7%), bicyclogermacrene (8.9%), germacrene D (7.6%), perilla aldehyde (3.5%) and β-caryophyllene (2.9%) were found to be the major constituents of the oil. The oil of the fruits of P. haussknechtii consisted of eight monoterpene hydrocarbons (1.7%), two oxygenated monoterpenes (3.9%), sixteen sesquiterpene hydrocarbons (26.8%), two oxygenated sesquiterpenes (2.1%) and five phenylpropanoids (58.7%). Three other nonterpenic compounds also comprised 1.7% of the oil.


Introduction
Umbelliferae family contains about 300 genera and 2500-3000 species distributed all around the world (1). The genus Pimpinella is one of the main genera of Umbelliferae and comprises more than 150 species (2). The genus represents in the flora of Iran by twenty species including six endemics (3). Previous phytochemical studies of Pimpinella species have led to the isolation of various compounds like phenylpropanoids (4) sesquiterpenes (5) coumarins (6) and volatile oils (7). According to pharmacological studies, the fruit of Anis (Pimpinella anisum) is widely used as carminative, expectorant and spasmolytic (8). It is also proved to possess antioxidant, antimicrobial, gastroprotective, antifungal, anticancer and bronchodilatory activities (9)(10)(11)(12)(13)(14). It is used in traditional medicine for menopausal hot flashes (14). In-vitro study of P. brachycarpa, edible greens grown in Asian regions, is found to have antioxidant effects (15). P. anisoides inhibits acetylcholinesterase (16) and presents protective Agricultural and Natural Resource Research Center. A voucher specimen (No 2827) was deposited at the Herbarium of the School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.

Extraction and isolation
The essential oil of the fruits of P. haussknechtii was obtained by hydrodistillation using a Clevenger-type apparatus for 3 h according to the method recommended by the British Pharmacopoeia (24). The volatile oil was dried over anhydrous sodium sulfate and stored in sealed vial at 4 °C until analysis. Gas chromatography combined with mass spectrometry was used for identification of the known oil components. Firstly, the analysis was performed on an Agilent 5975C mass selective detector coupled with an Agilent 7890A GC, equipped with an HP-5 GC capillary column (30 m × 0.25 mm; film thickness 0.25 μm). The oven temperature was programmed from 60-280 °C at the rate of 4°C per min. Helium was used as the carrier gas at a flow rate of 2 mL/min. Injector and detector temperatures were 280°C. The MS operating parameters were: ionization voltage, 70 eV; ion source temperature, 230°C; mass range, 35-425. The MSD ChemStation was used as operating software. Retention indices were calculated by using retention times of n-alkanes (C 8 -C 24 ) that were injected after the oil at the same conditions. Components of the oil were identified by comparison of their retention indices (RI) with those reported in the literature (21) and computer matching with NIST and Wiley275. L libraries. The fragmentation patterns of the mass spectra were also compared with those reported in the literature (25,27).
After GC-MS analysis, one unknown component (56.7%) was observed with retention time of 22.8 min, not characterized in the GC-MS library. In order to identify this compound, the essential oil was subjected on HPLC using YMC-Pak-Sil column (250 × 20 mm) with gradient system of hexane (A), and hexane: ethyl acetate, 9:1 (B) starting with A: B (100:0) for 20 min, then 0−20% B in 50 min, A:B (80:20) for 50 min, then 20-30% B in 30 min, and 30-100% B for 50 min. The flow rate was 3 mL/min, UV ditection at 210 and 270 nm , and the injection effect on oxidative damages (17). P. tirupatiensis have also shown cardio-protective activity on doxorubicin induced cardiotoxicity in rats (18). Economically, this genera are cultivated all around the world as medicinal plant. A few other species are cultivated for their aromatic fruits such as P. anisetum in Russia and P. saxifraga in India. P. peregrina and P. major are cultivated in Germany for their roots and P. calycina as vegetable (18). Pimpinella haussknechtii Rech. f. & Riedl (Syn. P. kotschyana Boiss.) (20) is an annual native plant which grows in the west of Iran.
Available information indicates that flavonoids and essential oils are two secondary metabolites which have been reported from different parts of P. kotschyana (22,21).
There are also a report on chemical composition and antimicrobial activity of P. kotschyana oil collected from Tehran province, Iran (23).
In this study, the volatile oil constituents of the friuts of P. haussknechtii grown in Lorestan province, Iran is reported by using the GC-MS analysis for the known components and high pressure liquid chromatography for unknown compound.

HPLC
(High-performance liquid chromatographic) analysis was done on a Waters system, equipped with 515 HPLC pump, and waters 2487 dual wavelenghth absorbance detector (Waters, Milford, MA, USA). The column was a YMC-Pak SIL (250 × 20 mm) (YMC Europe GmbH, Germany). The NMR spectra were recorded on a Bruker Avance AV 400 instrument, using CDCl 3 as a solvent. The IR spectrum was recorded on a Rayleigh WQF-510 FTIR spectrophotometer and the HREI-MS spectrum was measured in electron impact mode on Varian MAT 312 spectrometer.

Results and Discussion
More than thirty-six components were detected in the fruits of P. haussknechtii (Table 2). Thirty-five components of the oil were identified by GC-MS method and then HPLC method was used for the isolation of one unknown component (56.7%), which was not characterized in the GC-MS library with retention time of 22.8 min.
The fruits of P. haussknechtii yielded 1.5% (v/w) of yellowish oil with an aromatic odor. Essential oil yields of fruits of different Pimpinella species are very variable, for example the volatile oil yields of fruits of P. cretica var. arabica and P. isaurica are 10.0% and 0.3%, respectively. There are also other species that their fruits have no volatile oil (5).